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• W1965547460 abstract "The specific recognition mechanisms of DNA repair glycosylases that remove cationic alkylpurine bases in DNA are not well understood partly due to the absence of structures of these enzymes with their cognate bases. Here we report the solution structure of 3-methyladenine DNA glycosylase I (TAG) in complex with its 3-methyladenine (3-MeA) cognate base, and we have used chemical perturbation of the base in combination with mutagenesis of the enzyme to evaluate the role of hydrogen bonding and π-cation interactions in alkylated base recognition by this DNA repair enzyme. We find that TAG uses hydrogen bonding with heteroatoms on the base, van der Waals interactions with the 3-Me group, and conventional π-π stacking with a conserved Trp side chain to selectively bind neutral 3-MeA over the cationic form of the base. Discrimination against binding of the normal base adenine is derived from direct sensing of the 3-methyl group, leading to an induced-fit conformational change that engulfs the base in a box defined by five aromatic side chains. These findings indicate that base specific recognition by TAG does not involve strong π-cation interactions, and suggest a novel mechanism for alkylated base recognition and removal. The specific recognition mechanisms of DNA repair glycosylases that remove cationic alkylpurine bases in DNA are not well understood partly due to the absence of structures of these enzymes with their cognate bases. Here we report the solution structure of 3-methyladenine DNA glycosylase I (TAG) in complex with its 3-methyladenine (3-MeA) cognate base, and we have used chemical perturbation of the base in combination with mutagenesis of the enzyme to evaluate the role of hydrogen bonding and π-cation interactions in alkylated base recognition by this DNA repair enzyme. We find that TAG uses hydrogen bonding with heteroatoms on the base, van der Waals interactions with the 3-Me group, and conventional π-π stacking with a conserved Trp side chain to selectively bind neutral 3-MeA over the cationic form of the base. Discrimination against binding of the normal base adenine is derived from direct sensing of the 3-methyl group, leading to an induced-fit conformational change that engulfs the base in a box defined by five aromatic side chains. These findings indicate that base specific recognition by TAG does not involve strong π-cation interactions, and suggest a novel mechanism for alkylated base recognition and removal. DNA glycosylases are a remarkable enzyme class that recognize and remove damaged DNA bases as the first step in the DNA base excision repair pathway (1Mol C.D. Parikh S.S. Putnam C.D. Lo T.P. Tainer J.A. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 101-128Crossref PubMed Scopus (172) Google Scholar). This important pathway serves as the primary cellular defense against the accumulation of unwanted and toxic DNA base lesions (2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1278) Google Scholar). The removal of purine bases that have been alkylated at electronegative heteroatoms is the province of a highly specialized subgroup of these enzymes that specifically recognize and remove these unusual cationic DNA bases. One evolutionary solution to this problem in biological recognition is found in the human alkyladenine DNA glycosylase (AAG), 1The abbreviations used are: AAGalkyladenine DNA glycosylase3-MeA3-methyladenineTAG3-MeA DNA glycosylase IAlkA3-MeA DNA glycosylase IIHhHhelix-hairpin-helixNOEnuclear Overhauser effectNOESYNOE spectroscopyHPLChigh-performance liquid chromatography2Dtwo-dimensional3,7-DMA3,7-dimethyladenine3,9-DMA3,9-dimethyladeninewtwild type3,6-DMP3,6-dimethylpuriner.m.s.d.root mean square deviationHMQCheteronuclear multiple quantum coherence.1The abbreviations used are: AAGalkyladenine DNA glycosylase3-MeA3-methyladenineTAG3-MeA DNA glycosylase IAlkA3-MeA DNA glycosylase IIHhHhelix-hairpin-helixNOEnuclear Overhauser effectNOESYNOE spectroscopyHPLChigh-performance liquid chromatography2Dtwo-dimensional3,7-DMA3,7-dimethyladenine3,9-DMA3,9-dimethyladeninewtwild type3,6-DMP3,6-dimethylpuriner.m.s.d.root mean square deviationHMQCheteronuclear multiple quantum coherence. which has an α/β fold that is unique for DNA glycosylases (3Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar). However, many more alkylated purine-specific DNA glycosylases, such as 3-methyladenine (3-MeA) DNA glycosylase II (AlkA) from Escherichia coli, contain a highly conserved α-helical domain with a “helix-hairpin-helix” (HhH) DNA binding motif that is found in other DNA glycosylases and DNA-binding proteins (4Hollis T. Lau A. Ellenberger T. Mutat. Res. 2000; 460: 201-210Crossref PubMed Scopus (62) Google Scholar, 5Hollis T. Lau A. Ellenberger T. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 305-314Crossref PubMed Google Scholar). Both AAG and AlkA recognize a broad range of alkylated bases (3-methyladenine, 7-methyladenine, and 7-methylguanine), and the substrate range of AAG extends to nonalkylated bases (hypoxanthine and 1-N6-ethenoadenine) (4Hollis T. Lau A. Ellenberger T. Mutat. Res. 2000; 460: 201-210Crossref PubMed Scopus (62) Google Scholar, 6Asaeda A. Ide H. Asagoshi K. Matsuyama S. Tano K. Murakami A. Takamori Y. Kubo K. Biochemistry. 2000; 39: 1959-1965Crossref PubMed Scopus (50) Google Scholar). One recently described addition to the HhH DNA repair superfamily is 3-methyladenine DNA glycosylase I (TAG) from E. coli, which is unique in its high specificity for 3-MeA and 3-MeG (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). alkyladenine DNA glycosylase 3-methyladenine 3-MeA DNA glycosylase I 3-MeA DNA glycosylase II helix-hairpin-helix nuclear Overhauser effect NOE spectroscopy high-performance liquid chromatography two-dimensional 3,7-dimethyladenine 3,9-dimethyladenine wild type 3,6-dimethylpurine root mean square deviation heteronuclear multiple quantum coherence. alkyladenine DNA glycosylase 3-methyladenine 3-MeA DNA glycosylase I 3-MeA DNA glycosylase II helix-hairpin-helix nuclear Overhauser effect NOE spectroscopy high-performance liquid chromatography two-dimensional 3,7-dimethyladenine 3,9-dimethyladenine wild type 3,6-dimethylpurine root mean square deviation heteronuclear multiple quantum coherence. Enzymatic recognition and removal of cationic DNA bases such as 3-MeA represents a unique problem in DNA repair. However, repair of these lesions need not involve profound mechanisms for leaving group activation because these bases are electron-deficient and prone to spontaneous hydrolysis at 105- to 106-fold faster rates than neutral purine bases (8Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (696) Google Scholar, 9Stivers J.T. Jiang Y.L. Chem. Rev. 2003; 103: 2729-2760Crossref PubMed Scopus (396) Google Scholar). Consistent with this viewpoint, the active site pockets of AlkA and AAG possess no obvious polar groups capable of forming hydrogen bonds to the electronegative acceptor groups on the base to facilitate glycosidic bond cleavage, as might be expected from their abilities to remove a variety of bases (4Hollis T. Lau A. Ellenberger T. Mutat. Res. 2000; 460: 201-210Crossref PubMed Scopus (62) Google Scholar, 5Hollis T. Lau A. Ellenberger T. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 305-314Crossref PubMed Google Scholar). AlkA and AAG also possess no obvious binding pockets for the alkyl modification, presumably because of the relatively broad substrate specificity of these enzymes (10Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar). In fact, structural studies on AlkA and AAG revealed that their active sites are lined with conserved tryptophan and tyrosine residues that form stacking and edgewise interactions with the damaged cationic base (3Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar, 11Lau A.Y. Scharer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 12Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (203) Google Scholar). On the basis of the aromatic character of their active sites, it has been proposed that these enzymes use aromatic π-cation interactions to attract the cationic-damaged base into the active site, and thus discriminate between cationic-damaged purines and neutral undamaged purines (3Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar). Previous structural studies of TAG suggested both similarities and differences in base recognition and catalysis as compared with AAG and AlkA. From NMR chemical shift perturbation studies, the 3-MeA binding pocket of TAG was also assigned to an extremely aromatic-rich pocket, reminiscent of AAG and AlkA (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). However, TAG also has an absolutely conserved glutamic acid residue that was suggested to form hydrogen bonds with the N6 and N7 positions of 3-MeA in a similar fashion as an analogous group observed in the adenine binding pocket of MutY, an adenine-specific DNA glycosylase of the same superfamily (13Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (294) Google Scholar). In contradiction with expectations from the aromatic π-cation hypothesis, it was found that the neutral 3-methyladenine base bound specifically and tightly to TAG, whereas binding of the normal base adenine could not be detected (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). Thus, the TAG active site can specifically recognize a 3-methyladenine base even when its positive charge has been ablated, suggesting that other unique features of the base are detected by this enzyme. A deeper understanding of the catalytic mechanisms of alkylpurine-specific glycosylases is hampered by the absence of structural and mechanistic studies on the complexes of these enzymes with cationic and neutral alkylpurine bases. In this study we present the solution structure of TAG in complex with the neutral 3-MeA base. To complement the structural information, we have also synthesized a series of neutral and cationic 3-methyladenine base analogues to probe the role of charge and hydrogen bonding in specific base recognition. In addition, we have performed extensive mutagenesis of the enzyme to examine the energetic interactions of selected enzyme side chains with 3-MeA, as well as each of the 3-MeA base analogues. This comprehensive data set strongly indicates that TAG recognizes 3-MeA using specific hydrogen bonding, conventional π-π stacking interactions, and by van der Waals interactions with the 3-methyl group that lead to an induced fit conformational change in the enzyme. These results may provide evidence for a catalytic mechanism involving weak binding of the cationic substrate base in the ground state and tighter binding of the neutral base in the transition state. Sample Preparation—Samples of unlabeled and isotope-labeled TAG were prepared as described in Drohat et al. (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). The TAG mutants, Y13A, Y16A, H17A, D18A, W21A, E38A, and W46A were prepared using the QuikChange double-stranded mutagenesis kit from Stratagene (La Jolla, CA) using the pET28b overexpression vector, which includes an N-terminal His6 tag. All mutations were confirmed by DNA sequencing of both strands. The mutants were obtained in greater than 95% purity by sequential passage over nickel-agarose and SP-Sepharose columns. Enzyme concentrations were determined from UV absorbance measurements in 6 m guanidinium hydrochloride using calculated extinction coefficients (us.expasy.org/tools/protparam.html). For assignment of Trp and Tyr residues, and for the NOE experiments used to obtain restraints to the bound base, a uniformly 15N-labeled and fractionally deuterated wtTAG sample was prepared using minimal medium containing 75% 2H2O. Unlabeled tryptophan and tyrosine (0.2 g/liter) were added just prior to induction with isopropyl-1-thio-β-d-galactopyranoside to obtain selective labeling of these residues. For a typical NMR experiment, the purified protein was exchanged into NMR buffer (10 mm phosphate buffer, pH 6.6, 100 mm NaCl, 3 mm dithiothreitol, 0.34 mm NaN3), concentrated to 0.5–1 mm, and then 3-methyladenine was added to the solution to obtain a final concentration of about 5 mm. All NMR samples above were placed in Shigemi NMR tubes. Synthesis of Base Analogues—3-[8-13C]Methyladenine, 3,6-dimethylpurine, and 3,7-dimethyladenine were obtained by direct methylation of 8-[13C]adenine (Isotec), 6-methylpurine (Aldrich), and 3-methyladenine (Aldrich), respectively (14Fujii T. Saito T. Kizu K. Hayashibara H. Kumazawa Y. Nakajima S. Heterocycles. 1986; 24: 2449-2454Crossref Scopus (10) Google Scholar, 15Lam F.L. Parham J.C. Heterocycles. 1978; 9: 287-291Crossref Google Scholar, 16Ogilvie K.K. Beaucage S.L. Gillen M.F. Tetrahedron Lett. 1978; 35: 3203-3206Crossref Scopus (22) Google Scholar). Each of the reaction products was purified by reversed-phase HPLC, and the resultant compounds provided proton and 13C NMR spectra identical with previous reports (14Fujii T. Saito T. Kizu K. Hayashibara H. Kumazawa Y. Nakajima S. Heterocycles. 1986; 24: 2449-2454Crossref Scopus (10) Google Scholar, 15Lam F.L. Parham J.C. Heterocycles. 1978; 9: 287-291Crossref Google Scholar, 16Ogilvie K.K. Beaucage S.L. Gillen M.F. Tetrahedron Lett. 1978; 35: 3203-3206Crossref Scopus (22) Google Scholar). For the synthesis of 3,9-dimethyladenine, methyl iodide (70 mg) was added to 3-methyladenine (25.4 mg) in warm N,N-dimethylformamide (3 ml) and stirred for 30 min, followed by more methyl iodide (70 mg) with stirring overnight at room temperature. The precipitate was removed by centrifugation, and the clear solution was evaporated to dryness in vacuo. The final product was separated from other sideproducts by HPLC (10% overall yield). 1H NMR (D2O, ppm): δ 8.41 (s, 1H, 2-H); 8.14 (s, 1H, 8-H); 4.28 (s, 3H, 3-CH3), 4.16 (s, 3H, 9-CH3). UVmax: 203 and 269 nm. HRMS (matrix-assisted laser desorption ionization-Fourier transform mass spectroscopy) calc. for C7H10N5 (M + H), 164.093; found, 164.093. NMR Spectroscopy—NMR experiments were performed at 20 °C on Bruker DMX 500-, 600-, and 750-MHz NMR spectrometers, or Varian Unity Plus 600-MHz NMR spectrometers; all of which were equipped with four channels and pulse-field gradients. The standard suite of experiments for assigning 1H, 13C, and 15N backbone and side-chain chemical shifts and for obtaining NOE-based distance restraints were collected as previously described (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). Intermolecular NOE restraints between the 3-MeA base and 75% deuterated, 15N-labeled, and selectively Tyr-Trp protonated TAG (see above) were obtained using the 2D 1H-1H NOESY pulse sequence with a mixing time of 200 ms. Data were processed using NMRPipe (17Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11533) Google Scholar) and analyzed using SPARKY version 3 (T. D. Goddard and D. G. Kneller, University of California, San Francisco). The resonance assignments have been submitted to the BioMagResBank in Madison, WI (accession code 7834). The 1H chemical shifts were referenced to 2,2-dimethyl-2-silapentane-S-sulfonate, and the 13C and 15N resonances were indirectly referenced to 2,2-dimethyl-2-silapentane-S-sulfonate (18Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2068) Google Scholar). pKa of 3-MeA—To obtain the pKa value of the free and bound base, 2D 1H-13C HMQC experiments were collected using a Varian Unity Plus 500-MHz spectrometer for both free and TAG-bound 3-[8-13C]-methyladenine at several pH values. For each experiment sweep widths of 2514 Hz (13C) and 4999 Hz (1H) were used with the carbon and proton carriers set at 130 and 4.82 ppm, respectively. The sample of the complex contained 0.75 mm unlabeled TAG and 0.65 mm 3-[8-13C]-methyladenine in NMR buffer, such that about 95% of the base was enzyme-bound. Structural Calculations—Calculations were carried out as previously described (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). Interproton distance restraints obtained from NOESY experiments and backbone (φ/ψ) dihedral angle and hydrogen bond restraints derived from the observed 1Hα, 13CO, 13Cα, 13Cβ, and 15N chemical shifts, were used as inputs in the torsion angle dynamics simulated annealing protocol in CNS (19Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). A set of 200 structures was calculated, starting from a high quality CNS-derived structure, using the torsion angle dynamics simulated annealing protocol in the XPLOR-NIH program, 2G. M. Clore, J. Kuszewski, C. D. Schwieters, and N. Tjandra, XPLOR-NIH, version 2.0.2, nmr.cit.nih.gov/xplor-nih. including a torsion angle data base of mean potential force (20Kuszewski J. Gronenborn A.M. Clore G.M. Protein Sci. 1996; 5: 1067-1080Crossref PubMed Scopus (207) Google Scholar). From this set, 25 low energy structures were selected that exhibited no distance restraint violations of >0.5 Å and no dihedral angle restraint violations of >5.0° (Table I). After structural calculations using just intermolecular NOE restraints to the base, planar hydrogen bond restraints between the 3-MeA and the Oη-Hη of Tyr-16, and the Cδ-Oϵ1,ϵ2-Hϵ2 atoms of Glu-38 were then added as indicated by the initial structures and the biochemical data.Table IStructural statistics for 3-methyladenine DNA glycosylase I (TAG)Parameter{25}aThe 25 structures calculated and energy minimized in CNS.r.m.s.d. with respect to the mean structure Backbone atoms residues 11-1740.69 ± 0.10 All heavy atoms residues 11-1741.27 ± 0.14 Backbone atoms for all residues (1-188)1.04 ± 0.24 All heavy atoms for all residues (1-188)1.51 ± 0.19r.m.s.d. from experimental distance restraints (Å)bNone of the ensemble of structures displayed a distance violation of >0.5 Å. Intraresidue (365)0.050 ± 0.003 Sequential (706)0.035 ± 0.006 Medium range (604)0.027 ± 0.004 Long range (438)0.031 ± 0.004 Hydrogen bonds (132)0.044 ± 0.008 Intermolecular (27)0.108 ± 0.018r.m.s.d. from the ϕ/ψ dihedral angle restraints (222) (°)cBackbone torsion angle restraints were derived using TALOS (29). None of the structures exhibit a dihedral angle violation of >5°.0.45 ± 0.08r.m.s.d. from the experimental Cα chemical shifts1.34 ± 0.05r.m.s.d. from the experimental Cβ chemical shifts1.19 ± 0.02r.m.s.d. from idealized covalent geometry Bonds (Å)0.0028 ± 0.0002 Angles (°)0.497 ± 0.004 Impropers (°)0.419 ± 0.02Lennard-Jones potential energy (kcal·mol-1)dLennard-Jones potential energy calculated using the CHARMM parameters.↑ 699 ± 23Bad contacts per 100 residueseCalculated using the program PROCHECK (32).17.9 ± 2.8Ramachandran analysis (%)eCalculated using the program PROCHECK (32). Most favored86.1 ± 1.4 Additionally allowed9.6 ± 1.6 Generously allowed3.4 ± 0.9 Disallowed0.8 ± 0.5a The 25 structures calculated and energy minimized in CNS.b None of the ensemble of structures displayed a distance violation of >0.5 Å.c Backbone torsion angle restraints were derived using TALOS (29Parikh S.S. Walcher G. Jones G.D. Slupphaug G. Krokan H.E. Blackburn G.M. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5083-5088Crossref PubMed Scopus (244) Google Scholar). None of the structures exhibit a dihedral angle violation of >5°.d Lennard-Jones potential energy calculated using the CHARMM parameters.e Calculated using the program PROCHECK (32Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Fluorescence Spectroscopy—The binding of 3-MeA, 3,7-DMA, and 3,9-DMA to wtTAG and E38A was performed at 15 °C essentially as described in Drohat et al. (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). The melting curve profiles of TAG variants were obtained by monitoring the tryptophan fluorescence decrease of the enzymes with increasing temperature between 15 °C and 80 °C. The temperature was increased by 2 °C increments, and the samples were equilibrated for 1 min prior to measuring the fluorescence. A buffer containing 20 mm NaH2PO4, 100 mm NaCl (pH 7.5) was used, with excitation at 280 nm and fluorescence emission monitored at 343 nm using a Spex Fluoromax-3. Ultrafiltration Binding Assay—The binding affinities of wild-type and E38A for 3-MeA and 3,6-DMP were determined by ultrafiltration of the equilibrated mixture of enzyme and base analogue. The reaction mixtures (50 μl), consisting of 100 μm of each base analogue and a series of enzyme concentrations (0–550 μm), were incubated for 2 min and filtered for 5 min using microcon-3 (Millipore, Bedford, MA). The free base analogue in the filtrate was separated using a Phenomenex Aqua 5μ C-18 HPLC column using isocratic buffer elution (10 mm phosphate, 20% MeOH, pH 7). The 3-MeA peak area was then quantified by integration and normalized to an internal concentration standard consisting of 1 μm uracil that was included in the binding reaction. The equilibrium dissociation constants were obtained by nonlinear leastsquare analysis using Equation 1,[Complex]=[Base]tot×[E]free/(KD+[E]free)(Eq. 1) where Btotal and Efree represent the total concentration of base analogue and the free enzyme concentration, respectively. Solution Structure of the TAG Complex with 3-MeA—We have previously solved the solution structure of free TAG in the absence of any ligands (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). Here we have determined the solution structure of TAG in complex with the 3-MeA product using a similar suite of heteronuclear NMR experiments as previously employed to obtain the structure of the free enzyme (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar), and more recently, to refine the structure of the zinc binding site of TAG (21Kwon K. Cao C. Jiang Y.L. Stivers J.T. J. Biol. Chem. 2003; 278: 19442-19446Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). One special approach that was key to obtaining resonance assignments for the aromatic residues in the active site of the complex was a selective labeling experiment in which the 15N-labeled enzyme was partially deuterated by overexpression in 75% D2O, and just prior to induction, unlabeled Trp and Tyr were added to the minimal growth media to selectively protonate these groups. This approach reduced spin diffusion pathways and provided much more robust 2D NOESY spectra that allowed assignment of all key active site aromatic side chains and unambiguous measurements of intermolecular NOE values between these groups and the 3-MeA base. The overall quality of the structure for the complex is very similar to that of the free enzyme (r.m.s.d. = 0.64 Å, Fig. 1A), and the complete structural statistics are reported in Table I. Binding of the 3-MeA base (which is neutral at the pH of the NMR studies, see below) induces only small changes in the overall structure as compared with the free enzyme but alters the environment of Trp-6 (see below), giving rise to the large tryptophan fluorescence decrease that accompanies 3-MeA binding (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). A similar induced fit mechanism has been previously described for uracil DNA glycosylase upon binding to uracil (22Drohat A.C. Stivers J.T. Biochemistry. 2000; 39: 11865-11875Crossref PubMed Scopus (64) Google Scholar). Mapped onto the structure shown in Fig. 1B are the side chains of six active site amino acid residues that are completely conserved in all TAG family members (Trp-6, Tyr-13, Tyr-16, Trp-21, Glu-38, and Trp-46) (23Bujnicki J.M. Rychlewski L. DNA Repair (Amst.). 2002; 1: 391-395Crossref PubMed Scopus (6) Google Scholar). In the studies described below, these residues and two others, His-17 and Asp-18, were mutated to alanine. Structural Basis for 3-MeA Recognition—The position of the 3-MeA base in the active site was well defined by 27 intermolecular NOE restraints from the enzyme to the H8, H2, and 3-N-methyl hydrogens of the base. An overlay of the 3-MeA base and several surrounding residues that comprise the binding pocket, using the 10 lowest energy structures, is shown in Fig. 2A, and a molecular model of the lowest energy structure is shown in Fig. 2B. This structure reveals that the base is stacked with a single conserved tryptophan (Trp-46), and that Glu-38 serves as a bifunctional hydrogen bond donor and acceptor to N7 and the 6-amino group of 3-MeA. The hydroxyl of Tyr-16 is positioned to donate a hydrogen bond to the N1 position of the base, and Trp-6 appears to serve as a lid to encapsulate the 3-MeA base in an aromatic box defined by itself and Tyr-13, Tyr-16, and Trp-46 (Fig. 2B). Additional compelling evidence for a hydrogen bond between Glu-38 and the exocyclic amino group of 3-MeA is provided by the observation of a broad proton resonance in the complex with a chemical shift of δ (1H) = 6.13 ppm, which was unambiguously assigned to the 6-amino protons of 3-MeA in NOESY spectra. This combination of polar and π stacking interactions between 3-MeA and TAG differs considerably from the proposed aromatic π-cation recognition mechanisms of other alkylpurine DNA glycosylases such as AlkA and AAG (see above). Our previous finding that TAG could discriminate exquisitely between neutral 3-MeA and adenine suggested that the enzyme could sense the one difference between these bases: the 3-methyl group. As shown in the molecular surface representation of Fig. 2C, the methyl group is nestled in a pocket defined by the aromatic rings of Trp-6, Tyr-13, and the side chain methylene group of Trp-46. Favorable van der Waals interactions of the methyl group in this nonpolar pocket may be used to drive the induced-fit clamping of the enzyme, which creates the aromatic box around the 3-MeA base. Indeed, it is difficult to envision binding of 3-MeA in this aromatic box without it first being presented in an open conformation. The structure of free TAG is consistent with this idea, because the Trp-6 lid is “ajar,” allowing improved access of 3-MeA to the binding pocket (6Asaeda A. Ide H. Asagoshi K. Matsuyama S. Tano K. Murakami A. Takamori Y. Kubo K. Biochemistry. 2000; 39: 1959-1965Crossref PubMed Scopus (50) Google Scholar). This induced-fit mechanism involving nonpolar interactions with the 3-methyl group may explain the exceptional discrimination against adenine. Two other groups shown in Fig. 2C that are in van der Waals contact with 3-MeA are Ala-168 and Ser-164. Both of these groups are highly conserved in the TAG family, as would be expected from their close proximity to the bound base. The Bound 3-MeA Is Neutral—We have previously shown that TAG recognizes neutral 3-MeA, but not adenine, suggesting that positive charge was not required for specific binding (7Drohat A.C. Kwon K. Krosky D.J. Stivers J.T. Nat. Struct. Biol. 2002; 9: 659-664Crossref PubMed Scopus (54) Google Scholar). One caveat to this previous conclusion was that it could not be excluded that the enzyme protonated the neutral base upon binding, and that specific recognition did indeed involve favorable π-cation interactions between the enzyme and base. To directly address this question we incorporated a site-specific 13C label at the 8-carbon of the purine ring of 3-MeA and determined the pKa value of the free base using a 2D 1H-13C HMQC experiment (Fig. 3A). From this experiment, the pKa (3-MeA) = 5.6 ± 0.1, which is 1.9 log units lower than the pH value employed in the previous 3-MeA binding studies. To establish that 3-MeA remained neutral upon binding to the TAG active site, we measured the 8-13C shift of 3-MeA while it was bound in the active site pocket at pH 6.5 and 7.5 (Fig. 3B). The chemical shifts at both pH values were similar to free 3-MeA (Fig. 3A, closed circles), providing strong evidence that the enzyme does not significantly change the pKa value of the base, and that the 3-MeA binding measurements at pH 7.5 reflect binding of the neutral base. These findings establish the previous conclusion that specific recognition of 3-MeA need not involve positive charge on the base. 3An issue that is beyond the scope of this report is the tautomeric state of 3-MeA in DNA and in the TAG active site. Small molecule crystallographic studies of 3-methyl-2′-deoxyadenosine (30Fujii T. Saito T. Date T. Chem. Pharm. Bull. 1989; 37" @default.
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